Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 November - 17 December 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The LLNA method was not available at the time the study was conducted. Since an appropriate guinea pig maximisation test is available it can not be justified to conduct an additional LLNA study due to animal welfare reasons.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Test material: Simazine a.i.
- Appearance: White powder
- Lot No.of test material: 74
- Expiration date of the batch: September 1996
- Purity: 97.7%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the dark at 4°C

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: D. Hall, Newchurch, Staffordshire, England
- Microbiological status of animals, when known:
- Age at study initiation: 4 - 5 weeks
- Weight at study initiation: 286 - 366 g
- Housing: Groups of 5 animals in suspended metal cages with wire mesh floors
- Diet: Vitamin C enriched guinea-pig diet FD2 available ad libitum. Hay was given weekly.
- Water: available ad libitum
- Acclimation period: 12 days
- Indication of any skin lesions:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C
- Humidity (%): 30 - 70%
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12 hours light (0700 - 1900) : 12 hours dark

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
coconut oil
Concentration / amount:
10% w/v
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
coconut oil
Concentration / amount:
50% w/v
Day(s)/duration:
48 hours
Challenge
Route:
epicutaneous, occlusive
Vehicle:
coconut oil
Concentration / amount:
50% and 25% w/v
Day(s)/duration:
24 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Control: 5 animals
Test animals: 10 animals
Details on study design:
The test substance was prepared prior to each application on the day of dosing in Alembicol D (a product of coconut oil).

RANGE FINDING TESTS:
A preliminary study was performed to identify where possible:
a). concentrations of the test substance that would produce irritation suitable for the induction phase of the main study: The test material was administered by intradermal injections at concentrations of 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5 and 10.0 %w/v in Alembicol D in 2 animals.
b). a maximum non-irritant concentration by the topical route of administration for the challenge phase: The test material was administered by topical application at concentrations of 10.0, 20, 30 and 50 %w/v in Alembicol D in 4 animals.

MAIN STUDY
A. INDUCTION EXPOSURE - INTRADERMAL INJECTIONS:
- No. of exposures: 2 replicates of each injectable per animal (i.e. applied to both left and right injection sites); 10 animals tested
- Test groups: Injectables for the test animals were prepared as follows:
1. Freund's complete adjuvant was diluted with an equal volume of water for irrigation.
2. Simazine a.i., 10% w/v in Alembicol D
3. Simazine a.i., 10% w/v in a 50:50 mixture of Freund's complete adjuvant and Alembicol D
A volume of 0.1 mL was injected into both the left and right injection sites.
- Control group: 5 control animals were treated similarly to the test animals and were injected with the following test solutions:
1. Freund's complete adjuvant was diluted with an equal volume of water for irrigation.
2. Alembicol D
3. Freund's complete adjuvant 50:50 with Alembicol D
- Site: A 40 x 60 mm area of dorsal skin on the scapular region of the guinea-pig was clipped free of hair with electric clippers. Three pairs of intradermal injections were made into a 20 x 40 mm area within the clipped area.
- Concentrations: 10% w/v in Alembicol D

A. INDUCTION EXPOSURE - TOPICAL APPLICATIONS:
- No. of exposures: 2 replicates of each solution (i.e. applied to both left and right sites); 10 animals tested
- Exposure period: 48 hours
- Test groups: Treated with approximately 0.4 mL of Simazine a.i., 50% w/v in Alembicol D.
- Control group: 5 control animals were treated similarly to the test animals with the following solutions:
1. Freund's complete adjuvant was diluted with an equal volume of water for irrigation.
2. Alembicol D
3. Freund's complete adjuvant 50:50 with Alembicol D
- Site: Six days after the injections, the same 40 x 60 mm interscapular area was clipped and shaved free of hair and the site was pre-treated by gentle rubbing with 0.2 mL per site of 10% w/w sodium lauryl sulphate in petrolatum. 24 hours later a 20 x 40 mm patch of paper was saturated with approximately 0.4 mL of Simazine a.i., 50% w/v in Alembicol D. The patch was placed on the skin of the test animals and covered by a length of impermeable plastic adhesive tape. This in turn was firmly secured by elastic adhesive bandage wound around the torso of the animal and fixed with "Sleek" impervious plastic adhesive tape.
- Duration: The dressing was left in place for 48 hours.
- Concentrations: 50% w/v in Alembicol D

B. CHALLENGE EXPOSURE - TOPICAL APPLICATIONS:
- No. of exposures: 10 animals tested
- Time after induction: The control and test animals were challenged topically two weeks after the topical induction application.
- Day(s) of challenge: 72 hours after removal of the patches
- Exposure period: 24 hours
- Test groups: Treated with approximately 0.4 mL of Simazine a.i., 50% w/v in Alembicol D.
- Control group: 5 control animals were treated in an identical manner to the test animals.
- Site: Hair was removed by clipping and then shaving from an area on the left flank of each guinea pig. A 20 x 20 mm patch of paper was saturated with approximately 0.2 mL of Simazine a.i., 50% w/v in Alembicol D and applied to an anterior site on the flank. Simazine a.i., 25% w/v in Alembicol D was applied in a similar manner to a posterior site. The patches were sealed to the flank for 24 hours under strips of impermeable plastic adhesive tape covered by elastic adhesive bandage wound round the trunk and secured with "Sleek" impervious plastic adhesive tape.
- Concentrations: 50 and 25% w/v in Alembicol D
- Evaluation (hr after challenge): The challenge sites were evaluated 24, 48 and 72 hours after removal of the patches.
Challenge controls:
The challenge control animals were treated in the same way as the animals treated with the test material.
Positive control substance(s):
yes
Remarks:
Hexyl cinnamic aldehyde was used periodically to check the sensitivity of the guinea-pig strain.

Results and discussion

Positive control results:
The positive control results were valid.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50% w/v
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Localised dermal reaction
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
50% w/v
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Dryness and sloughing of the epidermis in one animal
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
72
Group:
test group
Dose level:
50% w/v
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Dryness and sloughing of the epidermis in one animal
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25% w/v
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Localised dermal reaction
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
25% w/v
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Dryness and sloughing of the epidermis in four animals
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
72
Group:
test group
Dose level:
25% w/v
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Dryness and sloughing of the epidermis in four animals
Remarks on result:
no indication of skin sensitisation

Any other information on results incl. tables

CLINICAL SIGNS:

No signs of ill health or toxicity were recorded.

BODYWEIGHT:

Bodyweight increases were recorded for all guinea-pigs over the period of the study.

INDUCTION:

Intradermal injections: Necrosis was recorded at sites receiving Freund's Complete Adjuvant in test and control animals. Slight irritation was seen in test animals at sites receiving Simazine a.i., 10% w/v in Alembicol D and slight irritation was observed in control animals receiving Alembicol D alone.

Topical application: Moderate erythema was observed in test animals following topical application with Simazine a.i. 50% w/v in Alembicol D and slight erythema was seen in the control guinea-pigs.

CHALLENGE:

The reactions seen in nine of the ten test animals were not definitely more marked than those seen in the controls, only isolated localised slight reactions were seen for three animals. The remaining animal gave an inconclusive response.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this study, Simazine a.i. did not produce evidence of skin sensitisation (delayed contact hypersensitivity) in nine of the ten test animals. The remaining animal gave an inconclusive response.